We have attacked the problem of DNA replication in mammalian nuclei by developing two in vitro systems that convert SV40 replicating DNA into mature, covalently closed, superhelical DNA; nuclei isolated from SV40 infected cells and a nuclei free nuclear extract containing SV40 nucleoprotein complexes. Our studies have shown that four fractions are required for viral DNA replication: an SV40 nucleoprotein complex containing SV40 replicating DNA, cytosol, nucleoplasmic fraction and an assay mix containing the four deoxynucleoside triphosphates, ATP, regenerating system, buffer, salts and a reducing agent. Cytosol is required for sustained DNA synthesis and to join "Okazaki pieces" to growing daughter strands. These fractions will be further fractionated and characterized. DNA replication in the reconstituted system will be analyzed for the distribution and fate of replicating DNA molecules, the initiation, elongation and termination of "Okazaki pieces" and the functional interactions between factors required for DNA replication. The results will then be compared with abortive forms of replication that occur when one or more of the required factors are missing in the cytosol depleted nuclei and SV40 replicating nucleoprotein complexes. Using this concept of in vitro complementation, we expect to correlate specific blocks in DNA replication with the function of specific factors. The final experiments will try to develop an in vitro system for studying the initiation of DNA replication in new viral genomes.